Diagnosis, monitoring, and treatment of conditions characterized by intracellular free radicals

Abstract

The present invention features the use of coelenterazine or a close analogue as a systemically administered real-time reporter of ROS or RNS levels in the environment of animal cells and organs. This real-time measurement can then be used to assess the severity of neurodegenerative diseases, ischemia-reperfusion injury after organ transplantation, acute radiation syndrome injury, and many other free radical-dependent disease states. The method can include real-time measurement of the therapeutic effect of PrC-210 as a free radical scavenger capable of detoxifying both oxygen and nitrogen free radicals.

Description

[Background technology]

[0001] The present invention features the use of coelenterazine or its analogs as a systematically administered real-time reporter of ROS or RNS levels in the cellular and organ environments of animals and humans. This real-time measurement can then be used to assess the severity of neurodegenerative diseases, ischemia-reperfusion injury after organ transplantation, acute radiation syndrome injury, and many other free radical-dependent disease states. The method can include real-time measurement of the therapeutic effect of PrC-210 as a free radical scavenger capable of detoxifying both oxygen and nitrogen free radicals. Summary of the Invention

[0002] In a first aspect, the invention features a method for measuring intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising: (i) administering to a subject a compound of formula (I),

number

[0003] In some embodiments, the method further comprises correlating the results of step (ii) to intracellular ROS and RNS levels in the subject.

[0004] In certain embodiments, the method further comprises using the results of step (ii) to monitor the onset or severity of the disease or condition in the subject.

[0005] In a related aspect, the invention features a method of treating a disease or condition mediated by intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising: (a) measuring intracellular ROS and RNS levels in a subject; (b) administering to the subject an effective amount of a compound of formula (II) or a pharmaceutically acceptable acid addition salt thereof based on the level measured in step (a); [ka] wherein (x) A is -CHNHR' and B is -CHNHR, or A = -NRR' and B = H; (y) each of R and R' is independently selected from H, C1-C6 alkyl, and C1-C6 heteroalkyl; With the proviso that when B=H, R and R′ are not both H. Step (a) can include performing a diagnostic method of the invention to measure the levels of intracellular ROS and RNS in the subject.

[0006] In certain embodiments, the compound of formula (II) is [ka] or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments of any of the above methods, the subject has received a solid organ transplant (SOT) or a vascularized composite allograft (VCA), and the transplant can be monitored for acute or chronic transplant rejection and therapeutic success.

[0008] In some embodiments of any of the above methods, the subject suffers from a neurodegenerative disease, including, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0009] In certain embodiments of any of the above methods, the subject suffers from multiple sclerosis or a neuroinflammatory disease, including, but not limited to, aseptic and non-aseptic encephalitis.

[0010] In certain embodiments of any of the above methods, the subject has suffered from an acute brain injury, such as a traumatic brain injury, spinal cord injury, or stroke.

[0011] In certain embodiments of any of the above methods, the subject suffers from acute radiation sickness or has been, is being, or will be exposed to low-energy and high-energy radiation from a nuclear explosion, a nuclear reactor leak, or space travel.

[0012] In some embodiments of any of the above methods, the subject suffers from a neuropsychiatric disorder, including, but not limited to, bipolar disorder (BD), schizophrenia, depression, anxiety disorder, attention deficit disorder, addiction, personality disorder, autism, and Asperger's syndrome.

[0013] In some embodiments of any of the above methods, the subject suffers from a cardiovascular disease, including, but not limited to, arteriosclerosis, myocardial infarction, angioplasty, heart valve disease, angina pectoris, peripheral circulatory disorders, vascular damage due to dialysis, vascular dementia, and transient ischemic attack.

[0014] In certain embodiments of any of the above methods, the subject suffers from a myopathic disease.

[0015] In some embodiments of any of the above methods, the subject suffers from an autoimmune disease, including, but not limited to, rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, and psoriasis.

[0016] In some embodiments of any of the above methods, the subject has type 2 diabetes, obesity, and metabolic syndrome.

[0017] In some embodiments of any of the above methods, the subject suffers from aging and age-related diseases.

[0018] In some embodiments of any of the above methods, the subject suffers from asthma and an inflammatory lung disease (eg, chronic obstructive pulmonary disease (COPD)).

[0019] In some embodiments of any of the above methods, the subject has suffered from acute trauma, including but not limited to, major trauma, major surgery, burns, and the like.

[0020] In some embodiments of any of the above methods, the subject is suffering from a complication of an infectious disease, including but not limited to, post-COVID syndrome.

[0021] In some embodiments of any of the above methods, the subject is suffering from a disease that leads to fibrosis, including, but not limited to, hepatitis that leads to liver fibrosis, glomerulonephritis that leads to kidney fibrosis, and primary and secondary pulmonary fibrosis.

[0022] In one particular embodiment of any of the above methods, the bioluminescence of step (ii) occurs in the absence of any luciferase enzyme.

[0023] definition To facilitate understanding of the present invention, a number of terms are defined below. Terms defined herein have meanings commonly understood by one of ordinary skill in the art relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer solely to a singular entity, but rather encompass the general type for which a specific example may be used for illustration. While terms herein are used to describe particular embodiments of the present invention, their use does not limit the invention except as defined in the claims.

[0024] As used herein, the term "about" refers to a value that is within 10% above or below the stated value.

[0025] As used herein, any value provided in a range of values ​​includes both the upper and lower limits, and any value subsumed within those limits.

[0026] As used herein, the term "pharmaceutically acceptable salt" refers to those salts of the described compounds that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, etc., within the normal scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977, and Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. These salts may be acid addition salts, including inorganic or organic acids. Salts may be prepared in situ during the final isolation and purification of the compounds described herein, or separately, by reacting the free base group with a suitable acid.

[0027] As used herein, the term "effective amount" refers to an amount sufficient to achieve a beneficial or desired result, such as a diagnostic result that determines disease activity. Therefore, determining a "therapeutically effective amount" depends on the context in which it is applied. For example, in the context of administering a luciferin precursor to provide a real-time report of intracellular ROS or RNS levels in a subject. The method may include real-time measurement of the therapeutic effect of PrC-210 as a free radical scavenger capable of detoxifying both oxygen and nitrogen free radicals. Thus, an effective amount of an aminothiol compound is, for example, an amount sufficient to ameliorate the symptoms or progression of a disease characterized by intracellular ROS and RNS species. Depending on the nature of the condition being treated, the method of the present invention may involve systemic (e.g., intravenous) or local administration (e.g., topical application or local injection) of the aminothiol compound.

[0028] As used herein, and as is well understood in the art, "diagnosing," "monitoring" disease activity, "treating" a condition, or "treatment" of various diseases or disorders is an effort to achieve a beneficial or desired result, e.g., a clinical outcome. Beneficial or desired results may include, but are not limited to, alleviation of one or more symptoms or conditions; a reduction in the severity of the disease, disorder, or condition; a stable (i.e., not worsening) state of the disease, disorder, or condition; a delay or slowing of the progression of the disease, disorder, or condition; an improvement or palliation of the disease, disorder, or condition; and a remission, either detectable or undetectable (either partial or complete). "Alleviating" a disease, disorder, or condition means that the severity and / or undesirable clinical signs of the disease, disorder, or condition are reduced and / or the time course of progression is slowed or prolonged compared to the severity or time course in the absence of treatment.

[0029] As used herein, the term "subject" may be a human, a non-human primate, or other mammal, such as, but not limited to, a dog, cat, horse, cow, pig, goat, monkey, rat, mouse, and sheep. In a preferred embodiment, the subject is a human.

[0030] As used herein, the term "pharmaceutical composition" refers to an active compound formulated with one or more pharmaceutically acceptable excipients. In some embodiments, the compounds of the present invention are present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In certain embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those suitable for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, or capsules; and parenteral administration, such as parenteral administration by subcutaneous, intramuscular, or intravenous injection.

[0031] As used herein, the term "pharmaceutically acceptable excipient" refers to any inert ingredient (e.g., a vehicle capable of suspending or dissolving an active compound) that is biocompatible and suitable for administration to a subject. Representative excipients include, for example, anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes, humectants, emulsifiers, diluents, film-forming or coating agents, flavors, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or hydration water. Excipients include, but are not limited to, optionally substituted butylated hydroxytoluene (e.g., BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, optionally substituted hydroxypropylcellulose, optionally substituted hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch, stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. Those skilled in the art are familiar with the variety of agents and substances useful as excipients.

[0032] As used herein, the term "alkyl" refers to a branched or straight-chain monovalent saturated aliphatic radical that, when unsubstituted, contains only C and H. A monovalent alkyl group does not include any substituents on the alkyl group. For example, when an alkyl group is attached to a compound, the monovalent alkyl means that it is attached to the compound and does not include any additional substituents that may be present on the alkyl group. In some embodiments, an alkyl group can contain, for example, 1 to 6, 1 to 4, or 1 to 2 carbon atoms (e.g., C1-C6, C1-C4, or C1-C2). Examples include, but are not limited to, methyl, ethyl, isobutyl, sec-butyl, tert-butyl, 2-methylpropyl, and 2,2-dimethylpropyl.

[0033] As used herein, the term "C1-C6 heteroalkyl" refers to a branched or straight-chain monovalent saturated alkyl radical containing C, H, and 1 to 3 N atoms.

[0034] As used herein, the term "luciferin" refers to a type of light-emitting pigment found in organisms capable of bioluminescence, as well as synthetic analogs or functionally equivalent chemicals that are oxidized to produce oxyluciferin and light energy. D-luciferin, or 2-(6-hydroxybenzothiazol-2-yl)-2-thiazoline-4-carboxylic acid, was first isolated from the firefly Photinus pyralis. Various chemically distinct forms of luciferin have been discovered and studied, primarily in marine organisms such as fish and squid, but many have also been identified in terrestrial organisms, such as worms, beetles, and various other insects (Day et al. (2004) Luminescence 19:8-20). As used herein, luciferin also includes derivatives or analogs of luciferin. In addition to entirely synthetic luciferins, such as cycloalkylaminoluciferins (CycLucl), there are at least five general types of biologically evolved luciferins that are chemically distinct and catalyzed by different chemically and structurally distinct luciferases that use a variety of different cofactors. First, firefly luciferin, which is the substrate for firefly luciferase and requires ATP for catalysis (EC 1.13.12.7). Second, bacterial luciferin, also found in some squid and fish, consists of a long-chain aldehyde and reduced riboflavin phosphate. Bacterial luciferases are FMNH-dependent. Third, deinoflagellate luciferin, a tetrapyrrole-based chlorophyll derivative found in deinoflagellates (marine plankton), organisms responsible for nocturnal marine phosphorescence. Deinoflagellate luciferase catalyzes the oxidation of deinoflagellate luciferin and consists of three identical catalytically active domains. Fourth, there is bulgurin, an imidazolopyrazine found in certain mollusks and deep-sea fish, such as Polycytis. Finally, there is coelenterazine (an imidazolopyrazine), a light-emitting substance of the protein aequorin, found in radiolarians, crickets, cnidarians, squids, copepods, whales, fish, and shrimp.

[0035] As used herein, the term "coelenterazine" refers to a class of luciferins that function as substrates in cnidarians, krill, capillaries, sea slugs, decapod shrimp, daphnia, radiolarians, and some fish (Greer and Szalay (2002) Luminescence 17:43-74). For example, in the case of sea urchin luciferase, coelenterazine analogs / derivatives that produce light emission at 418-547 nm are available (Loeng et al. (2007) Nature Methods 4:641-643). Coelenterazine analogs / derivatives (400A, DeepBlueC) have been described that, together with Renilla luciferase, emit light at 400 nm (WO 01 / 46691, incorporated herein by reference). Other examples of coelenterazine luciferins are enduRen, Proloom Purple, Proloom Purple II, Proloom Purple III, viviRen, and furimazine. Other examples of coelenterazine analogs / derivatives include, but are not limited to, the compounds disclosed in U.S. Patent No. 9,624,425 and U.S. Patent Publication No. 20140302539, each of which is incorporated herein by reference.

[0036] Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims. [Brief explanation of the drawings]

[0037] [Figure 1] The chemical reaction of photon emission from coelenterazine is shown. [Figure 2] 1 shows a comparison of the chemical structures of coelenterazine, enduRen, and vivRen. [Figure 3] Comparison of the biodistribution between injected coelenterazine and ViviRen in mice. Coelenterazine induces photon signals primarily within blood vessels when injected intravenously, rather than in specific organ locations. ViviRen results in photon emissions evenly distributed throughout the body. [Figure 4]Panels a and b show the treatment of U373 MG human astrocytoma cells in tissue culture with phorbol myristate (PMA). Addition of PMA to the culture medium induced superoxide production (measured by reaction with coelenterazine). In panel b, PrC-210 was observed to scavenge and suppress (reduce to background levels) the generated superoxide in a dose-dependent manner. Panel c shows the PMA dose-dependent superoxide production (measured by reaction with ViviRen) in previously irradiated mice. The data show that intraperitoneal injection of PrC-210 (0.5 MTD, 252 mg / kg bw) prior to PMA administration significantly reduced detectable levels of superoxide in mice. [Figure 5] 1 shows a comparison of the biodistribution of viviRen in mice following intravenous and subcutaneous injection. [Figure 6] 1 shows imaged ROS levels according to organ- and time-specific distribution patterns after irradiation with 8.68 Gy. [Figure 7] (A) Free radical levels in mice after 8.68 Gy irradiation are suppressed to background levels by systemically administered PrC-210. (B,C) Levels of caspase 3 / 7 markers in mouse plasma after 8.68 Gy irradiation. This biomarker is elevated without PrC-210 treatment and suppressed to background levels by PrC-210: (B) 0.5 MTD of PrC-210 administered 30 min before 8.68 Gy irradiation or (C) 0.3 MTD of PrC-210 administered 24 h after irradiation. [Figure 8] Shown are the levels of ROS (A), MDA (B), and caspase-1 (C) in the brain of mice after 8.68 Gy irradiation; ROS and MDA levels are suppressed to background levels by systemically administered PrC-210. [Figure 9]We show the levels of ROS, caspase 8, and caspase 3 / 7 markers in mouse brains after 8.68 Gy of irradiation; these biomarkers are elevated without treatment and suppressed to background levels by PrC-210. Caspase levels "reflect" ROS levels. [Figure 10] (A) Quantitative measurement of mouse brain ROS levels (measured by reaction with IV ViviRen) in three SOD1G93A mutant mice and wild-type controls. (B) Body weight over time for the same three SOD1G93A mice. With the onset of ALS motor symptoms and hind limb paralysis, weight loss progressed, correlating with the increased ROS levels in (A). Systemic administration of PrC-210 (0.1 MTD, IP) was associated with an immediate plateau of weight loss. [Figure 11] Whole body free radical levels after irradiation with different radiation doses are shown, either without PrC-210 or with A) different amounts of PrC-210 administered intravenously 30 minutes before irradiation and free radical imaging 30 minutes after irradiation, or B) different amounts of PrC-210 administered intravenously just before free radical imaging 30 minutes after irradiation. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention features the use of a luciferin precursor administered to provide a real-time reporter of ROS and RNS levels in the environment of animal and human cells and organs. This real-time measurement can then be used to assess the activity and severity of neurodegenerative diseases, post-organ transplant ischemia-reperfusion injury, acute radiation syndrome injury, and many other free radical-dependent disease states. The method can include real-time measurement of the therapeutic effect of PrC-210 as a free radical scavenger capable of detoxifying both oxygen and nitrogen free radicals. Luciferin and luciferin precursors

[0039] The method of the present invention features the use of the bioluminescent luciferin coelenterazine (Shimomura O, Johnson FH, "Chemical Properties of the Bioluminescence System in Marine Animals," Proceedings of the National Academy of Sciences of the United States of America, April 1975), which emits one photon when oxidized by a free radical molecule (Figure 1). The emitted photons can be quantitatively measured using a bioluminescence monitor, such as a Lago X, spectral instrument (https: / / spectralinvivo.com / imaging-systems / ). The light-emitting oxidation reaction of coelenterazine does not require the presence of an additional catalytic enzyme. Therefore, coelenterazine can react and emit a photon whenever and wherever it comes into contact with a free radical species. Coelenterazine does not depend on an enzyme, such as luciferase, to be oxidized and emit a photon. This enzyme-independent reaction makes luciferin derivatives such as coelenterazine a stand-alone diagnostic method for detecting free radicals in situ.

[0040] Coelenterazine refers to a class of luciferins that function as substrates in cnidarians, krill, capillaries, sea slugs, decapod shrimp, daphnia, radiolarians, and some fish (Greer and Szalay (2002) Luminescence 17:43-74). For example, in the case of sea urchin luciferase, coelenterazine analogs / derivatives that produce light emission at 418-547 nm are available (Loeng et al. (2007) Nature Methods 4:641-643). Coelenterazine analogs / derivatives (400A, DeepBlueC) have been described to emit light at 400 nm with Renilla luciferase (WO 01 / 46691, incorporated herein by reference). Other examples of coelenterazine luciferins are enduRen, Proloom Purple, Proloom Purple II, Proloom Purple III, viviRen, and furimazine. Other examples of coelenterazine analogs / derivatives useful in the methods of the present invention include, but are not limited to, the compounds disclosed in U.S. Pat. No. 9,624,425 and U.S. Patent Publication No. 20140302539, each of which is incorporated herein by reference.

[0041] Other luciferin compounds, such as firefly luciferin and vargine, can be used in place of coelenterazine. The present invention uses coelenterazine because it emits blue light, which allows for greater tissue penetration. This is important because the present invention is used to measure photons emitted from tissue sites in living organisms. Tissues and organs such as the brain, kidneys, heart, and intestines are located several millimeters below the body surface. The emitted blue light has a shorter wavelength, allowing it to penetrate body tissue more efficiently and therefore be detectable at the body surface. For example, vargine also emits blue light and can be used in place of coelenterazine. Furthermore, adding or removing benzyl rings can increase or decrease the rigidity of the compound or change its polarity, potentially shifting the emission wavelength to shorter (ultraviolet 380-10 nm) or longer (near-infrared 500-2500 nm) wavelengths depending on the detection needs. Administration of luciferin precursor

[0042] The animal and human dosage of the coelenterazine reporter of the present invention varies depending on factors including the route of administration, the disease being diagnosed or monitored and treated, the subject's physical characteristics, such as age, weight, and general health, the target organ, and the target species, such as humans, mice, or pigs. Generally, the dose of the coelenterazine reporter molecule (e.g., coelenterazine) disclosed herein can be contained in a single dose and be sufficient to distinguish free radical concentrations above background levels. The administered dose is adjusted by the clinician based on animal and human studies and is determined based on conventional factors such as the extent of the disease, the method and location of free radical measurement (e.g., whole-body photometric measurement, endoscopy, intraorgan sensors, transdermal or topical measurement), and various physiological parameters of the subject. It also depends on the safety margin of the compound, which may be 100-fold higher than the concentrations of currently used compounds. For example, the LD50 of viviRen orally administered to rats is 14,500 mg / kg, which is 100-fold higher than the dose used in our experiments. As shown in Figure 4, standard curves must be established for phorbol myristate (PMA)-treated U373 cells and irradiated ICR mice to demonstrate the background free radical concentration and therapeutic window for a specific species. In this model, PMA induces the generation of superoxide radicals, a free radical species with a very short half-life. Figure 2, panel A, shows PMA-induced superoxide radical generation in tissue culture of human U373 astroglioma cells, followed by PrC-210 concentration-dependent inhibition (scavenging) of the generated superoxide (B). U373 cells were cultured in DMEM medium containing 10% FBS, and PMA was added to the medium in a 10% ethanol:90% saline solution. After 30 seconds, coelenterazine was added to the medium, and coelenterazine photon emission was continuously monitored in an IVIS imaging chamber. PrC-210 was added to the medium 10 minutes before the addition of PMA. In panel C, PMA in a 20% ethanol:80% saline solution was injected IV into the tail vein of an ICR mouse. Ten minutes later, ViviRen was injected into the tail vein of the mouse.The levels of superoxide-ViviRen complexes in mice were monitored by quantifying photon emission in an IVIS chamber. As shown, IP injection of 0.5 MTD of PrC-210 (252 mg / kg) immediately prior to IV PMA administration to mice resulted in a significant reduction in measurable superoxide-ViviRen production. Measurement of intracellular ROS / RNS levels

[0043] The present invention further features methods for measuring organ and "disease-associated" free radical concentrations. Therefore, it may sometimes be necessary to ensure that only intracellular free radicals are measured. To ensure this, a luciferin repeater molecule, such as coelenterazine, must be linked to a cleavable blocking group that prevents oxidation and subsequent photon emission as long as the blocking group is attached to the luciferin. This blocking group must be selected so that it does not prevent the luciferin reporter from being taken up by cells or from crossing the blood-brain barrier. Good examples of these conjugated luciferins ("blocked luciferins") are viviRen and enduRen (Otto-Duessel M, Khankaldyyan V, Gonzalez-Gomez I, Jensen MC, Laug WE, Rosol M., "In Vivo Testing of Renilla Luciferase Substrate Analogs in an Orthotopic Murine Model of Human Glioblastoma," Molecular Imaging, 5(2), 2006), which are analogs of coelenterazine (Figure 2). The linked blocking group is cleaved by cellular lipases and esterases once the compound enters the cell. Enzymes endogenous to all cells are used as the cleavage mechanism for the blocking group cleavage. This cleavage then activates coelenterazine or its corresponding analog, which can be oxidized and emit photons. Coelenterazine without a blocking group structure is primarily oxidized at the point of administration or within the blood vessels before reaching the target organ (see example of coelenterazine in Figure 3). In the present invention, intracellular release is shown to lead to uniform distribution of luciferin throughout the body (see viviRen in Figure 3).

[0044] The photon signal emitted by the luciferin substrate generated by coelenterazine or its analogs is measured using a bioluminescence device, such as a Perkin-Elmer IVIS instrument. Other methods for measuring photons include charge-coupled devices (CCDs) or complementary metal-oxide semiconductor (CMOS) devices. Because the present invention does not rely on catalytic enzymes such as luciferase, the amount of measured fluorescence signal is significantly lower than that measured from mutant cells expressing these catalytic enzymes and therefore cannot be measured using standard measurement protocols. To receive sufficient photon signal for differential analysis, exposure times often require longer acquisition times. Acquisition times can potentially be shortened by: i) increasing the photon sensitivity of the bioluminescent device used for measurement; ii) increasing the amount of bioluminescent compound; or iii) optimizing the wavelength for a specific organ or measurement modality.

[0045] Potential imaging applications include:

[0046] Photon detection, either inside or outside the body, is accomplished by cameras capable of converting captured photons into electrons. For example, digital cameras, including DSLRs, mirrorless cameras, and smartphone cameras, use CCD (charge-coupled device) or CMOS (complementary metal-oxide semiconductor) sensors to capture images. Both CCD and CMOS sensors are used to detect light and convert it into an electrical signal, which is then processed into an image.

[0047] Skin measurements: A wearable device with a CCD (charge-coupled device) camera or smartphone camera-CMOS (complementary metal-oxide semiconductor) sensor for imaging through a thin dermal layer (e.g., wrist or finger) to determine overall inflammatory status (e.g., for monitoring Alzheimer's disease, multiple sclerosis (MS), rheumatoid arthritis, lupus, and vasculitis).

[0048] Subcutaneous measurements: Transcutaneous fiber optics connected to optical sensors or CCD cameras, similar to those used in continuous glucose monitoring devices, may be used to assess the overall inflammatory state in autoimmune diseases, e.g., multiple sclerosis (MS), rheumatism, arteriosclerotic conditions, aging, and / or neurodegeneration in Alzheimer's, ALS, or Parkinson's disease.

[0049] Intramuscular measurements: Needles for intramuscular imaging are known in the art to assess muscle degeneration (potential decrease in signal) or inflammation (increase in signal) (e.g., ALS, MS) (see, e.g., Sanchez, Gabriel N., et al., Neuron 88.6(2015):1109-1120).

[0050] Intra-organ measurements can be made using fiber optics placed within the organ connected to a CCD camera to measure inflammatory disease activity, for example in the brain, liver, kidney, pancreas, etc.

[0051] Imaging of hollow organs such as the digestive tract, respiratory system, nasal cavity, and kidneys can be performed using CCD scopes, including gastrointestinal imaging (e.g., routine or intraoperative laparoscopic imaging for the detection of tumors or inflammatory bowel disease), the respiratory system (e.g., bronchoalveolar imaging for inflammation), the nasal cavity with the possibility of deep brain imaging, e.g., the pituitary gland, corpus callosum from the nasal cavity, e.g., for Alzheimer's disease or ALS), imaging of the kidneys (ureteroscopy for renal disease), or arthroscopy to determine the state of arthritis.

[0052] Arterial imaging is imaging of arteries to determine the state of arteriosclerosis, or cardiac imaging to determine the state of myocarditis.

[0053] Aminothiol Aminothiols useful in the present process can be synthesized, for example, as described in US Pat. No. 7,314,959.

[0054] Aminothiol administration The dosage of the compounds disclosed herein depends on factors such as the route of administration, the disease to be treated, and the age, weight, and general health of the subject. Typically, the amount of the compounds disclosed herein (e.g., PrC-210) can be an amount that effectively treats the disease without causing serious toxicity, whether administered in a single dose or multiple doses over a long period of time. The dosage can be adjusted by a clinician based on conventional factors such as the extent of the disease and various parameters of the subject. Generally, the pharmaceutical compositions disclosed herein can be administered in an amount of about 0.001 mg to about 500 mg / kg / day of an aminothiol such as PrC-210.

[0055] For all species, a "normal" free radical range, dependent on age, skin pigmentation, and body weight, can be established, which serves as a standard of comparison for determining deviations of free radicals from the normal range (see, e.g., Curtis et al., Temporal variations of skin pigmentation in C57BL / 6 mice affect optical bioluminescence quantitation. Mol Imaging Biol. 2011;13(6):1114-1123.). Animal and human studies can be used to establish the amount and dosing schedule of PrC-210 required to keep free radicals within this normal range for each species and disease.

[0056] Pharmaceutical Compositions The pharmaceutical compositions of the present invention comprise one or more compounds disclosed herein (e.g., one or more compounds of Formula (I), Formula (IA), and Table 1) as therapeutic compounds. In addition to a therapeutically effective amount of the compound, the pharmaceutical compositions also contain a pharmaceutically acceptable excipient and can be formulated by methods known to those of skill in the art. In some embodiments, pharmaceutical compositions for treating disease contain one or more of the compounds disclosed herein (e.g., one or more compounds of Formula (I), Formula (IA), and Table 1), and can be formulated and / or administered with or without other therapeutic agents for a particular condition. Examples of such therapeutic agents (second therapeutic agents) are described herein.

[0057] The compounds disclosed herein (e.g., compounds of Formula (I), Formula (IA), and Table 1) can be used in the form of a free base or in the form of a salt, and as solvates. All forms are within the scope of the present disclosure.

[0058] Exemplary routes of administration of a pharmaceutical composition (or compound of the composition) include oral, sublingual, buccal, transdermal, intradermal, intramuscular, parenteral, intravenous, intraarterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, intranasal, inhalation, and topical administration.

[0059] treatment method In certain embodiments, the invention involves correlating intracellular bioluminescence with intracellular ROS and RNS levels in a subject. The measured levels can be used to monitor the etiology or severity of a disease or condition in the subject. The measured levels can also be used to determine the effective amount of an aminothiol (a compound of Formula (II), e.g., PrC-210) needed to treat ROS and RNS in a subject.

[0060] These methods are useful in treating, monitoring, and evaluating subjects undergoing solid organ transplantation (SOT) or vascularized composite allografts (VCA), and can monitor the transplant for acute or chronic transplant rejection and therapeutic success.

[0061] These methods can be useful in treating, monitoring, and assessing subjects suffering from neurodegenerative diseases, including, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0062] These methods can be useful in treating, monitoring, and evaluating subjects suffering from neuropsychiatric disorders, including, but not limited to, bipolar disorder (BD), schizophrenia, depression, anxiety disorders, attention deficit disorder, addiction, personality disorders, autism, and Asperger's syndrome.

[0063] These methods can be useful in treating, monitoring, and evaluating subjects suffering from cardiovascular diseases, including, but not limited to, arteriosclerosis, myocardial infarction, angioplasty, heart valve disease, angina pectoris, peripheral circulatory disorders, vascular damage due to dialysis, vascular dementia, and transient ischemic attacks.

[0064] These methods can be useful in treating, monitoring, and evaluating subjects suffering from neuroinflammatory diseases, including, but not limited to, multiple sclerosis or aseptic and non-aseptic encephalitis.

[0065] These methods can be useful in the treatment, monitoring, and evaluation of subjects suffering from acute brain injury, such as traumatic brain injury, spinal cord injury, or stroke.

[0066] These methods can be useful in treating, monitoring, and evaluating subjects suffering from myopathic disorders.

[0067] These methods can be useful in treating, monitoring, and evaluating subjects suffering from autoimmune diseases, including, but not limited to, rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, and psoriasis.

[0068] These methods can be useful in the treatment, monitoring, and evaluation of subjects suffering from type 2 diabetes, obesity, and metabolic syndrome.

[0069] These methods can be useful in treating, monitoring, and assessing subjects suffering from aging and age-related diseases.

[0070] These methods can be useful in treating, monitoring, and evaluating subjects suffering from asthma and inflammatory lung diseases (eg, chronic obstructive pulmonary disease (COPD)).

[0071] These methods can be useful in the treatment, monitoring, and evaluation of subjects suffering from acute trauma, including but not limited to, serious trauma, major surgery, and burns.

[0072] These methods can be useful in treating, monitoring, and evaluating subjects suffering from complications of infectious diseases, including but not limited to, post-COVID syndrome.

[0073] These methods can be useful in treating, monitoring, and evaluating subjects suffering from diseases that lead to fibrosis, including, but not limited to, hepatitis that leads to liver fibrosis, glomerulonephritis that leads to kidney fibrosis, and primary and secondary pulmonary fibrosis.

[0074] These methods can be useful in the treatment, monitoring, and evaluation of subjects suffering from acute radiation sickness or who have been exposed to, are being exposed to, or will be exposed to low-energy and high-energy radiation from a nuclear explosion, a nuclear reactor leak, or space travel.

[0075] The following examples are intended to illustrate the invention, but are not intended to limit the invention in any way. [Example]

[0076] Example 1. Continuous monitoring of free radical levels in vivo. ICR mice received 8.68 Gy of whole-body radiation at time "0." At subsequent times, ranging from 2 hours to 196 hours after radiation, as indicated at the bottom of the image (Figure 6), each mouse was anesthetized with 3% isoflurane and a bolus of ViviRen was administered intravenously into the tail vein. Subsequently, the mouse's whole-body ROS-ViviRen-generated photons were continuously monitored and quantified in an IVIS chamber. As seen in the image panels, free radical levels follow an organ-specific pattern over time. Interestingly, this pattern mirrors the time-dependent development of organ-specific toxicity after radiation exposure reported by the CDC. (See, for example, https: / / www.cdc.gov / nceh / radiation / emergencies / arsphysicianfactsheet.htm).

[0077] Example 2. Monitoring free radical levels after irradiation. ICR mice were irradiated with 8.68 Gy of whole-body radiation at time "0" (Figure 7). At the times indicated on the x-axis, individual mice were anesthetized with 3% isoflurane, and a bolus of ViviRen was injected IV into the mouse's tail vein. Subsequently, the mouse's whole-body ROS-ViviRen-generated photons were continuously monitored and quantified in an IVIS chamber. Treatment of mice with PrC-210, either i) at 0.5 MTD (252 mg / kg) 30 min before irradiation, or ii) at 0.3 MTD (151 mg / kg) 24 h after irradiation, significantly suppressed ROS signal intensity in intact mice. Interestingly, plasma caspase 3 / 7 levels mirrored the free radical concentrations seen in the IVIS chamber during the first 48 h after irradiation, and the markers remained completely suppressed to background for up to 6 days after PrC-210 treatment. Free radical levels follow the same pattern, but there is a peak after day 6, which is a sign of early multi-organ failure based on the organ pattern in Figure 6, involving the liver and lungs.

[0078] Example 3. Monitoring the reduction of free radical levels after irradiation and administration of PrC-210. To visualize the ROS response after irradiation, ICR mice were irradiated with 8.68 Gy of whole-body radiation at time point "0" (Figure 8). At the times indicated on the x-axis, individual mice were anesthetized with 3% isoflurane, and a bolus of ViviRen was injected IV into the mouse's tail vein. Subsequently, brain ROS-ViviRen-generated photons were continuously monitored and quantified in an IVIS chamber. Treatment of mice with PrC-210 at either i) 0.5 MTD (252 mg / kg) 30 minutes before irradiation or ii) 0.3 MTD (151 mg / kg) 24 hours after irradiation significantly suppressed ROS signal intensity in intact mice.

[0079] To measure the effects of radiation-induced ROS on the mouse brain, ICR mice were irradiated with 8.68 Gy of whole-body radiation (Figure 7). At the indicated times after irradiation, they were euthanized, and their brains were removed and homogenized in Tris buffer (pH 7.4). Homogenates were analyzed fresh or stored at -80°C before analysis. Malondialdehyde (MDA, a ROS-oxidized lipid) and caspase-1 (a free radical-induced inflammasome) levels in brain homogenates were measured as previously described in our study (Verhoeven, BM, Fahl, WE et al., Transplantation Direct 6:8 e578-586, 2020). MDA and caspase-1 levels were normalized to 100 μg of protein in the brain homogenates. Mice were intraperitoneally administered sodium chloride (control), PrC-210 (0.5 MTD, 252 mg / kg bw) 30 min before irradiation, or PrC-210 (0.3 MTD, 151 mg / kg) 24 h after irradiation. Analysis of MDA and caspase-1 was performed in triplicate. P values ​​for comparisons between PrC-210-treated and control groups are shown. Brain ROS levels with and without PrC-210 treatment correlated almost perfectly with MDA and caspase-1 levels over the entire experimental time period. Increases in malondialdehyde (MDA) as a marker of lipid peroxidation (Stefan Gawel, Maria Wardas, Elzbieta Niedworok, Piotr Wardas, Wiad Lek, 2004;57(9-10):453-5) and caspase-1 in secondary responses to increased free radicals (Fabio Martinon, ROS Signaling Promotes Inflammasome Activation, European Journal of Immunology, Volume 40, Issue 3, March 2010, Pages 616-61) are both a result of increased free radical levels and oxidative stress.

[0080] Example 4. Capase levels track free radical levels after irradiation and PrC-210 therapy. Similar to Example 3, 4ICR mice were irradiated with 8.68 Gy of whole-body radiation at time "0" (Figure 8). At the times indicated on the X-axis, individual mice were anesthetized with 3% isoflurane, and a bolus of ViviRen was injected IV into the tail vein of the mouse. Subsequently, mouse brain ROS-ViviRen-generated photons were continuously monitored and quantified in an IVIS chamber. Treatment of mice with PrC-210 at either i) 0.5 MTD (252 mg / kg) 30 minutes before irradiation or ii) 0.3 MTD (151 mg / kg) 24 hours after irradiation significantly suppressed ROS signal intensity in intact mice. Unlike Example 3, caspase-8 and caspase-3 / 7 were analyzed in brain samples collected at different times indicated on the X-axis. There were two main findings from this experiment: i) activation of caspase-8 and caspase-3 / 7 reflected ROS levels with a slight delay, which makes sense because the apoptotic pathway needs to be activated first in response to oxidative stress caused by free radicals, and ii) free radical levels were suppressed in the same way after PrC-210 treatment, following a similar suppression pattern in caspase-8 and caspase-3 / 7 activity over the duration of the experiment.

[0081] Example 5. Continuous monitoring of free radical levels in brain cells in vivo. This experiment was designed to clarify the relationship between brain free radical levels and the onset and progression of neurodegenerative diseases such as amyotrophic lateral sclerosis. G93A Quantitative measurements of ROS levels (measured by reaction with IV ViviRen) in the brains of mutant and wild-type control mice are shown. Figure 10, Panel B, shows the same three mice as in Panel A, SOD1 G93A The weight loss of mice was progressive and exponential with the onset of ALS motor symptoms.

[0082] There were two main findings from this experiment: i) the timing of onset of motor symptoms in three mice directly correlated with increased levels of free radicals in three mice, and ii) administration of systemic PrC-210 (0.1 MTD, IP) was associated with immediate stabilization of body weight and cessation of weight loss. Example 6. Relationship between radiation dose and free radical levels, and measurement of free radical scavenging by PrC-210.

[0083] This experiment was designed to investigate (i) the relationship between different radiation doses given to mice and the free radical levels generated in their organs as a result of radiation exposure, and (ii) the ability of PrC-210 to suppress radiation-induced free radical levels in mouse organs through its scavenging ability.

[0084] Figures 11A and B show that there is a linear relationship between radiation dose and free radical levels measured with viviRen 30 minutes after irradiation.

[0085] FIG. 11A shows that PrC-210 injected intravenously 30 minutes before irradiation reduces free radical levels in a PrC-210 dose-dependent manner.

[0086] Both Figures A and B show that increasing doses of PrC-210 suppress free radicals to background levels as the dose of radiation increases, meaning that it is possible to define for any radiation dose the dose of PrC-210 at which radiation-induced free radicals are suppressed to background levels, regardless of whether PrC-210 is administered before or after irradiation.

[0087] Panel B shows that PrC-210 reduces radiation-induced free radicals in a dose-dependent manner.

[0088] Both panels of Figure 11 show a linear relationship between the administered radiation dose and organ free radical production as measured by viviRen. The panels also show a dose-dependent relationship between radiation-induced free radical levels and their suppression by PrC-210 dose. These data can be used i) to measure the biological effects of specific radiation doses on cells / organs by measuring free radical levels in cells / organs using viviRen, and ii) to measure the free radical scavenging effect of PrC-210 on the suppression of free radical levels, regardless of whether PrC-210 was administered before or after irradiation.

[0089] Other embodiments Various modifications and variations of the described compositions, methods, and uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the present invention.

[0090] Other embodiments are within the scope of the following claims.

Claims

1. 1. A method for measuring intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising: (i) administering to said subject a compound of formula (I), [Equation 1] wherein A is a radical of luciferin, each O is an oxygen atom connecting A and B, each B is an independently cleavable protecting group, and n is an integer from 1 to 4; (ii) following step (i), measuring intracellular bioluminescence from the luciferin in vitro.

2. 2. The method of claim 1, wherein A is a radical of coelenterazine, valgrine, prolume purple, or furimazine.

3. The method of claim 2, wherein A is a radical of coelenterazine.

4. The method of claim 3 , wherein the radical of the coelenterazine is enduRen or viviRen.

5. The method of any one of claims 1 to 4, wherein each cleavable protecting group B is selected to be cleaved by an esterase or a lipase.

6. 6. The method of any one of claims 1 to 5, further comprising correlating the results of step (ii) with intracellular ROS and RNS levels in the subject.

7. 6. The method of any one of claims 1 to 5, further comprising using the results of step (ii) to monitor the onset or severity of a disease or condition in said subject.

8. 1. A method of treating a disease or condition mediated by intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in a subject, the method comprising: (a) measuring intracellular ROS and RNS levels in said subject; (b) administering to the subject an effective amount of a compound of formula (II) or a pharmaceutically acceptable acid addition salt thereof based on the level measured in step (a); 【Chemistry 1】 During the ceremony, (x) A is -CH ２ NHR' and B is -CH ２ NHR, or A=-NRR' and B=H; (y) each of R and R′ is independently H, C １ ~C ６ Alkyl, and C １ ~C ６ heteroalkyl; The method, wherein when B=H, then R and R′ are not both H.

9. The method of claim 8, wherein step (a) comprises carrying out a method according to any one of claims 1 to 7.

10. The compound of formula (II) is 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

11. 11. The method of any one of claims 1 to 10, wherein the subject has undergone a solid organ transplant (SOT) or a vascularized composite allograft (VCA), and the transplant can be monitored for acute or chronic transplant rejection and therapeutic success.

12. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from a neurodegenerative disease, including but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

13. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from a neuroinflammatory disease, including but not limited to multiple sclerosis or aseptic and non-aseptic encephalitis.

14. The method of any one of claims 1 to 10, wherein the subject has suffered from an acute brain injury, such as a traumatic brain injury, a spinal cord injury, or a stroke.

15. 11. The method of any one of claims 1 to 10, wherein the subject suffers from acute radiation sickness or has been exposed to, is being exposed to, or will be exposed to low-energy and high-energy radiation from a nuclear explosion, a nuclear reactor leak, or space travel.

16. 11. The method of any one of claims 1 to 10, wherein the subject suffers from a neuropsychiatric disorder, including, but not limited to, bipolar disorder (BD), schizophrenia, depression, anxiety disorder, attention deficit disorder, addiction, personality disorder, autism, and Asperger's syndrome.

17. 11. The method of any one of claims 1 to 10, wherein the subject suffers from a cardiovascular disease, including, but not limited to, arteriosclerosis, myocardial infarction, angioplasty, heart valve disease, angina pectoris, peripheral circulatory disorders, vascular damage due to dialysis, vascular dementia, and transient ischemic attack.

18. The method of any one of claims 1 to 10, wherein the subject suffers from a myopathic disease.

19. 11. The method of any one of claims 1 to 10, wherein the subject suffers from an autoimmune disease, including but not limited to rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, and psoriasis.

20. The method of any one of claims 1 to 10, wherein the subject suffers from type 2 diabetes, obesity, and metabolic syndrome.

21. The method of any one of claims 1 to 10, wherein the subject suffers from aging and age-related diseases.

22. The method of any one of claims 1 to 10, wherein the subject suffers from asthma and an inflammatory lung disease (e.g., chronic obstructive pulmonary disease (COPD)).

23. 11. The method of any one of claims 1 to 10, wherein the subject has suffered from acute trauma, including but not limited to, major trauma, major surgery, and burns.

24. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from a complication of an infectious disease, including but not limited to, post-COVID syndrome.

25. 11. The method of any one of claims 1 to 10, wherein the subject is suffering from a disease that leads to fibrosis, including, but not limited to, hepatitis that leads to liver fibrosis, glomerulonephritis that leads to kidney fibrosis, and primary and secondary pulmonary fibrosis.

26. 11. The method of any one of claims 1 to 10, wherein the bioluminescence in step (ii) occurs in the absence of any luciferase enzyme.

Images